Assessment of metal-reducing bacteria residing in industrial wastewater: a bacterial-based bioremediation approach

Tehmina Sajid; Ropahla Aslam; Abeer Kazmi; Aleeza Kazmi; Saad farooqi; Afifa Tariq; Ali Muhammad

doi:10.15419/br.v8i1.787

Research articles

Assessment of metal-reducing bacteria residing in industrial wastewater: a bacterial-based bioremediation approach

Tehmina Sajid ¹

Ropahla Aslam ²

Abeer Kazmi ^{3, 4, *}

Aleeza Kazmi ⁵

Saad farooqi ⁶

Afifa Tariq ¹

Ali Muhammad ⁷

Department of Chemistry, Lahore College for Women University, Lahore, Pakistan
Department of Chemistry, University of Management and Technology (UMT), Lahore, Pakistan
Department of Biotechnology, Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan (AWKUM), Mardan, Pakistan
Department of Genetics, Institute of Hydrobiology, University of Chinese Academy of Sciences (UCAS), Wuhan,PR China
Department of Microbiology, Shaheed Benazir Bhutto Women University (SBBWU), Peshawar, Pakistan
Department of Environmental Sciences, Institute of Hydrobiology, University of Chinese Academy of Sciences (UCAS), Wuhan,PR China
The Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, China

Correspondence to: Abeer Kazmi, Department of Biotechnology, Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan (AWKUM), Mardan, Pakistan; Department of Genetics, Institute of Hydrobiology, University of Chinese Academy of Sciences (UCAS), Wuhan,PR China. Email: [email protected].

Volume & Issue: Vol. 8 No. 1 (2022) | Page No.: Article ID 787 | DOI: 10.15419/br.v8i1.787

Published: 2022-03-31

Abstract

Background: Metals are required in minor amounts in the human body to perform vital functions, but beyond that level, they become toxic and cause many harmful effects. At present, polluted water is a large source of these heavy metals inside our body. Bioremediation is one of best treatments for the removal of these heavy metals from water. Objectives: The main objective of the current study was to isolate metal-reducing bacteria from soil and wastewater samples from different industries. Furthermore, the metal reducing potential of the bacteria was also evaluated under various environmental conditions (pH, temperature, incubation time, and UV exposure).

Methodology: Different bacterial strains were isolated that were resistant to different concentrations of zinc (Zn) and chromium (Cr) from wastewater and soil samples from four different industries (Riaz textile mills, Sitara chemical industry, Mandiali paper mills and Siddique leather works). Morphological characterization was carried out with the help of Gram staining, spore staining and motility tests. Biochemical tests were performed, such as catalase, oxidase, and H2S production tests, starch hydrolysis tests and Simon citrate tests. Chromium reduction after UV exposure was calculated to check mutation effects on chromium reduction.

Results: A total of 14 bacterial strains were isolated from the soil and wastewater samples. Six strains were Zn resistant, while eight strains were chromium resistant. A total of five strains were isolated from wastewater out of fourteen strains, while the remaining nine strains were isolated from soil samples of these industries. The bacterial strains were rod-shaped cocci and coccobacillus. The growth of bacterial strains under different environmental conditions, such as temperature, pH and incubation time, was observed, and the best growth was found at 37◦C, pH 7.0 and after 48 hrs. Maximum reduction at different concentrations was observed at pH 7.0 and 37◦C and after 48 hrs.

Conclusion: In conclusion, the bacterial strains isolated from industrial wastes and soils showed significant resistivity against various concentrations of Zn and Cr and reduced it efficiently under different conditions (pH, incubation time, temperature and UV light). The use of microorganisms for bioremediation is an environmentally friendly and cost-effective approach to reduce heavy metals present in our water and soil.

Keywords: Heavy metals Chromium Bioremediation Bacterial Strains

INTRODUCTION

Heavy metals are defined as metals with a particular density greater than 5 g/cm that have a negative impact on the environment and living beings. When present in extremely low quantities, these metals are important for maintaining different biochemical and physiological processes in living organisms; nevertheless, when concentrations surpass specific thresholds, they become toxic 1, 2. Heavy metals are emitted by natural and anthropogenic activities into the environment. Most heavy metals reach the environment from mining operations3. Heavy metals also persist in the environment even when mining activities are stopped. This was due to anthropogenic activities such as different foods, cosmetics and other chemicals that we used in our routines contain heavy metals 4. Heavy metals are present in the waste of many industries, such as the tanning, textile and electroplating industries5. When added to other water resources, including drinking water, their wastewater becomes a cause of toxicity 6. Water contaminated with these heavy metals when applied to agricultural areas enters into food crops through the water uptake of plants7. Due to their nonbiodegradable nature, heavy metals enter the food chain and affect living species8. When we consume food with heavy metals, they enter the human body and affect bodily functions, causing disease depending upon the metal. The most frequent heavy metals detected in waste water are arsenic, cadmium, chromium, copper, lead, nickel, and zinc, all of which pose health and environmental hazards9.

Chromium pollution of the environment, specifically hexavalent chromium pollution, has been a major problem in recent years10. Cr(III) is oxidized to Cr(VI) in the presence of sufficient oxygen in the environment, which is incredibly poisonous and readily soluble in water11. Excess chromium in the environment is harmful to plants because it alters the biological components of the plant and enters the food chain via the ingestion of these plant materials7, 12. Reduced root development, leaf chlorosis, germination rate inhibition, and low biomass are all common symptoms of Cr phytotoxicity13, 14, 15. Chromium (VI) has corrosive properties and can induce allergic responses in the body. As a result, inhaling excessive quantities of chromium (VI) can irritate the nasal lining and lead to nose ulcers16. It can also harm sperm and the male reproductive system17, causing anemia18, irritations, and ulcers in the small intestine and stomach19. Chromium also induces allergic responses, including significant skin redness and edema20. Humans may have serious cardiovascular, pulmonary, hematological, gastrointestinal, renal, hepatic, and neurological consequences, as well as mortality, when exposed to excessively high concentrations of chromium(VI) compounds16, 21, 22.

Several technologies have been developed for the treatment of water containing heavy metals and other waste. Different synthetic compounds are used to treat such waste types, but they are too expensive. One and only effective and cost-effective method is the biodegradation of heavy metals from bacteria. The main objective of the current study was to isolate these bacteria from soil and wastewater samples from different industries, including the leather industry, paper mill, chemical industry and textile industry, which have the potential to reduce and degrade chromium (Cr) at different temperatures, pH values and incubation times. For bacterial characterization, different morphological, biochemical and physiological tests were also performed. The effect of DNA mutation on chromium reduction was also observed after exposure to UV.

METHODOLOGY

Sample collection

Samples were collected from different industries, including Siddique leather works, Mandiali paper mills, Sitara chemical industry and Riaz textile mills. Waste water and soil samples of these four industries were collected by using a bottle following safety precautions. These samples were stored in properly labeled bottles. These samples were stored under proper conditions before laboratory experiments to avoid reactions (Table 1).

Table 1

Soil and wastewater samples from different industries

Sr.no	Samples	Industries
1	Sample no 1 (L1)	Siddique leather works
2	Sample no 2 (L2)	Mandiali paper mills
3	Sample no 3 (L3)	Sitara chemical industries
4	Sample no 4 (L4)	Riaz textile mills

Table 2

Zinc and Chromium resistant bacterial strains isolated from different industrial samples

Sr.no	Strains	Samples
1	S₁ZnL₁	Soil sample of Siddique leather works
2	S₂ZnL₁	Soil sample of Siddique leather works
3	S₃ZnL₁	Soil sample of Siddique leather works
4	S₄ZnL₁	Soil sample of Siddique leather works
5	S₅ZnL₃	Soil sample of Sitara chemical industries
6	W₁ZnL₁	Water sample of Siddique leather works
7	S₂CrL₂(A)	Soil sample of Mandiali paper mills
8	S₂CrL₂(B)	Soil sample of Mandiali paper mills
9	W₁CrL₃(A)	Water sample of sitara chemical industries
10	W₁CrL₃(B)	Water sample of sitara chemical industries
11	S₄CrL₄	Soil sample of Riaz textile mills
12	W₄CrL₄	Water sample of Riaz textile mills
13	W₃CrL₂	Water sample of Mandiali paper mills
14	S₅CrL₁	Soil sample of Siddique textile mills

Table 3

Colony morphology of zinc (Zn)- and chromium (Cr)-resistant strains

Sr.no	Strains	Colony morphology
		Size	Form	Pigment	Margin	Elevation	Texture	Opacity
1	S₁ZnL₁	Large	Circular	Cream	Entire	Convex	Mucoid	Non- opaque
2	S₂ZnL₁	Medium	Irregular	Off white	Entire	Umbonate	Shiny	Opaque
3	S₃ZnL₁	Medium	Circular	Off white	Entire	Flat	Shiny	Non- opaque
4	S₄ZnL₁	Medium	Circular	Yellow	Entire	Flat	Shiny	Non- opaque
5	S₅ZnL₃	Large	Circular	Cream	Entire	Raised	Mucoid	Non- opaque
6	W₁ZnL₁	Large	Circular	Off white	Entire	Flat	Shiny	Non- opaque
7	S₂CrL₂(A)	Medium	Circular	Cream	Entire	Raised	Mucoid	Non- opaque
8	S₂CrL₂(B)	Small	Irregular	White	Lobate	Umbonate	Rough	Non- opaque
9	W₁CrL₃(A)	Medium	Circular	White	Entire	Flat	Mucoid	Non- opaque
10	W₁CrL₃(B)	Medium	Circular	Off white	Entire	Flat	Mucoid	Non- opaque
11	S₄CrL₄	Small	Circular	Cream	Entire	Convex	Mucoid	Non- opaque
12	W₄CrL₄	Small	Circular	Yellow	Entire	Umbonate	Shiny	Opaque
13	W₃CrL₂	Medium	Circular	Off white	Entire	Flat	Mucoid	Non- opaque
14	S₅CrL₁	Large	Rhizoid	White	Filifom	crateriform	Rough	Non- opaque

Table 4

Cell morphology of zinc (Zn)- and chromium (Cr)-resistant strains

Sr.no	Strains	Cell shape	Gram Staining	Spore formation	Motility
1	S₁ZnL₁	Coccus	+	+	-
2	S₂ZnL₁	Rod	+	+	-
3	S₃ZnL₁	Rod	-	+	-
4	S₄ZnL₁	Coccus	+	+	-
5	S₅ZnL₃	Coccobacilli	+	+	-
6	W₁ZnL₁	Rod	+	+	-
7	S₂CrL₂(A)	Rod	+	+	-
8	S₂CrL₂(B)	Coccus	+	+	-
9	W₁CrL₃(A)	Coccus	+	+	-
10	W₁CrL₃(B)	Coccus	+	+	-
11	S₄CrL₄	Coccobacilli	+	+	-
12	W₄CrL₄	Rod	+	+	-
13	W₃CrL₂	Rod	+	+	-
14	S₅CrL₁	Rod	-	+	-

Table 5

Biochemical characterization of isolated bacterial strains

Sr.no	Strains	Catalase	Oxidase	Indole test	H2S production test	Starch Hydrolysis test
1	S₁ZnL₁	+	+	-	-	-
2	S₂ZnL₁	+	+	-	-	-
3	S₃ZnL₁	+	+	-	-	-
4	S₄ZnL₁	+	+	-	-	+
5	S₅ZnL3	+	+	-	-	+
6	W₁ZnL₁	+	+	-	-	+
7	S₂CrL₂(A)	+	+	-	-	-
8	S₂CrL₂(B)	+	+	-	-	+
9	W₁CrL₃(A)	+	+	-	-	+
10	W₁CrL₃(B)	+	+	-	-	-
11	S₄CrL₄	+	+	-	-	+
12	W₄CrL₄	+	+	-	-	+
13	W₃CrL₂	+	+	-	-	-
14	S₅CrL₁	+	+	-	-	-

Table 6

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 100 µl concentration

Sr no	Strains	Cross Metal resistance at 100µl concentration
		Zinc (Zn)	Chromium (Cr)	Cobalt (Co)	Manganese (Mn)	Nickle (Ni)	Silicon (Si)	Selenium (Se)
1	S₁ZnL₁	o	o	o	o	o	×	o
2	S₂ZnL₁	o	o	o	o	o	×	o
3	S₃ZnL₁	o	×	o	o	o	×	o
4	S₄ZnL₁	o	o	o	o	o	o	o
5	S₅ZnL₃	o	o	o	o	o	×	o
6	W₁ZnL₁	o	o	o	o	o	o	o
7	S₂CrL₂(A)	o	o	o	o	o	×	o
8	S₂CrL₂(B)	×	o	o	o	o	o	o
9	W₁CrL₃ (A)	o	o	×	o	o	×	o
10	W₁CrL₃ (B)	o	o	×	o	o	o	o
11	S₄CrL₄	o	o	o	o	o	×	o
12	W₄CrL₄	o	o	o	o	o	×	o
13	W₃CrL₂	o	o	o	o	o	×	o
14	S₅CrL₁	o	o	×	o	o	o	o

Table 7

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 150 µl

Sr no	Strains	Cross Metal resistance at 150µl concentration
		Zinc (Zn)	Chromium (Cr)	Cobalt (Co)	Manganese (Mn)	Nickle (Ni)	Silicon (Si)	Selenium (Se)
1	S₁ZnL₁	o	o	o	o	o	×	o
2	S₂ZnL₁	o	o	o	o	o	×	o
3	S₃ZnL₁	o	×	o	o	o	×	o
4	S₄ZnL₁	o	o	o	o	o	o	o
5	S₅ZnL₃	o	o	o	o	o	×	o
6	W₁ZnL₁	o	×	o	o	o	o	o
7	S₂CrL₂(A)	o	o	o	o	o	×	o
8	S₂CrL₂(B)	×	o	o	o	o	×	o
9	W₁CrL₃ (A)	o	o	×	o	o	×	o
10	W₁CrL₃ (B)	o	o	×	o	o	×	o
11	S₄CrL₄	o	o	×	o	o	×	o
12	W₄CrL₄	o	o	o	o	o	×	o
13	W₃CrL₂	o	o	o	o	o	×	o
14	S₅CrL₁	×	o	×	o	o	o	o

Table 8

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 200 µl

Sr.n o	Strains	Cross Metal resistance at 200µl concentration
Sr.n o	Strains	Zinc (Zn)	Chromium (Cr)	Cobalt (Co)	Manganese (Mn)	Nickle (Ni)	Silicon (Si)	Selenium(Se)
1	S1ZnL1	o	o	o	o	o	×	o
2	S2ZnL1	o	o	o	o	o	×	o
3	S3ZnL1	o	×	o	o	×	×	o
4	S4ZnL1	o	o	o	o	o	o	o
5	S5ZnL3	o	o	×	o	o	×	o
6	W1ZnL1	o	×	o	o	o	o	o
7	S2CrL2 (A)	o	o	o	o	o	×	o
8	S2CrL2 (B)	×	o	o	o	o	×	o
9	W1CrL3 (A)	o	o	×	o	o	×	o
10	W1CrL3 (B)	×	o	×	o	×	×	o
11	S4CrL4	×	o	×	o	o	×	o
12	W4CrL4	o	o	o	o	o	×	o
13	W3CrL2	×	o	o	o	o	×	o
14	S5CrL1	×	o	×	o	×	o	o

Table 9

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 300 µl

Sr no	Strains	Cross Metal resistance at 300µl concentration
Sr no	Strains	Zinc (Zn)	Chromium (Cr)	Cobalt (Co)	Manganese (Mn)	Nickle (Ni)	Silicon (Si)	Selenium (Se)
1	S₁ZnL₁	o	o	o	o	×	×	o
2	S₂ZnL₁	o	o	o	o	×	×	o
3	S₃ZnL₁	o	×	o	o	×	×	o
4	S₄ZnL₁	o	o	o	o	×	o	o
5	S₅ZnL₃	o	o	×	o	×	×	o
6	W₁ZnL₁	o	×	o	×	×	o	o
7	S₂CrL₂ (A)	o	o	o	o	×	×	o
8	S₂CrL₂ (B)	×	o	o	o	×	×	o
9	W₁CrL₃ (A)	×	o	×	o	×	×	o
10	W₁CrL₃ (B)	×	o	×	o	×	×	o
11	S₄CrL₄	×	o	×	o	o	×	o
12	W₄CrL₄	o	o	o	o	×	×	o
13	W₃CrL₂	×	o	o	o	×	×	o
14	S₅CrL₁	×	o	×	o	×	o	o

Table 10

Growth of zinc (Zn)- and chromium (Cr)-resistant strains after different time intervals

Sr. no	Strains	Time (hours)
Sr. no	Strains	2	4	6	18	24	48
1	S₁ZnL₁	0.041	0.472	0.561	1.89	1.989	2.000
2	S₂ZnL₁	0.058	0.530	0.676	1.213	1.24	0.949
3	S₃ZnL₁	0.031	0.452	0.613	0.921	1.924	1.543
4	S₄ZnL₁	0.059	0.563	0.721	0.986	1.014	1.986
5	S₅ZnL₃	0.050	0.439	0.689	0.890	0.909	1.893
6	W₁ZnL₁	0.030	0.621	0.798	0.940	0.953	1.923
7	S₂CrL₂ (A)	0.073	0.534	0.890	1.231	1.240	2.100
8	S₂CrL₂ (B)	0.045	0.567	0.888	0.987	0.990	1.712
9	W₁CrL₃ (A)	0.035	0.478	0.650	0.989	1.001	1.754
10	W₁CrL₃ (B)	0.023	0.211	0.567	0.811	0.813	1.432
11	S₄CrL₄	0.041	0.314	0.659	0.989	0.990	1.976
12	W₄CrL₄	0.121	0.362	0.789	0.912	0.930	2.213
13	W₃CrL₂	0.090	0.450	0.889	0.995	0.100	2.221
14	S₅CrL₁	0.061	0.313	0.540	0.777	0.780	1.653

Table 11

Effect of pH on the growth of zinc (Zn)- and chromium (Cr)-resistant strains

Sr. no	Strains	Optical Density at Different Ph (O.D)
Sr. no	Strains	3	5	9
1	S₁ZnL₁	0.412	1.226	0.728
2	S₂ZnL₁	0.451	1.162	0.577
3	S₃ZnL₁	0.460	0.944	0.633
4	S₄ZnL₁	0.409	1.200	0.409
5	S₅ZnL₃	0.375	1.052	0.721
6	W₁ZnL₁	0.286	0.796	1.100
7	S₂CrL₂(A)	0.688	1.209	0.488
8	S₂CrL₂(B)	0.241	0.472	0.644
9	W₁CrL₃(A)	0.245	0.312	0.503
10	W₁CrL₃(B)	0.441	1.078	0.618
11	S₄CrL₄	0.406	0.671	0.204
12	W₄CrL₄	0.280	0.521	0.423
13	W₃CrL₂	0.277	0.705	0.570
14	S₅CrL₁	0.185	1.179	0.223

Table 12

Effect of temperature on the growth of zinc (Zn)- and chromium (Cr)-resistant strains

Sr. no	Strains	Optical Density at Different Temperatures (O.D)
Sr. no	Strains	27^oC	37^oC	20^oC	42^oC
1	S₁ZnL₁	0.22	0.752	0.466	0.604
2	S₂ZnL₁	0.946	0.637	0.303	0.412
3	S₃ZnL₁	1.808	0.616	0.316	1.109
4	S₄ZnL₁	0.191	1.522	0.456	0.709
5	S₅ZnL₃	1.602	1.368	0.535	0.654
6	W₁ZnL₁	0.671	1.098	0.589	0.809
7	S₂CrL₂(A)	0.954	1.140	0.649	0.840
8	S₂CrL₂(B)	0.507	0.883	0.439	0.228
9	W₁CrL₃(A)	0.610	1.218	0.283	0.246
10	W₁CrL₃(B)	0.482	1.370	0.375	1.062
11	S₄CrL₄	0.201	1.368	0.322	0.267
12	W₄CrL₄	0.747	1.848	0.300	0.364
13	W₃CrL₂	0.588	1.694	0.297	0.367
14	S₅CrL₁	0.279	0.980	0.329	0.255

Table 13

Effect of incubation time on the growth of zinc (Zn)- and chromium (Cr)-resistant strains

Sr. no	Strains	Optical Density at Different incubation time at 37oC (O.D)
Sr. no	Strains	24hrs	48hrs	72hrs
1	S₁ZnL₁	0.752	1.011	1.272
2	S₂ZnL₁	0.637	0.926	1.110
3	S₃ZnL₁	0.616	0.879	1.314
4	S₄ZnL₁	1.522	1.721	1.897
5	S₅ZnL₃	1.368	1.509	1.627
6	W₁ZnL₁	1.098	1.143	1.204
7	S₂CrL₂(A)	1.140	1.532	1.619
8	S₂CrL₂(B)	0.883	1.090	1.213

Biotechnological Research

Assessment of metal-reducing bacteria residing in industrial wastewater: a bacterial-based bioremediation approach

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Abstract

INTRODUCTION

METHODOLOGY

Sample collection